N-glycosylation in Archaea-New roles for an ancient posttranslational modification.

Genome analysis points to N-glycosylation as being an almost universal posttranslational modification in Archaea. Although such predictions have been confirmed in only a limited number of species, such studies are making it increasingly clear that the N-linked glycans which decorate archaeal glycoproteins present diversity in terms of both glycan composition and architecture far beyond what is seen in the other two domains of life. In addition to continuing to decipher pathways of N-glycosylation, recent efforts have revealed how Archaea exploit this variability in novel roles. As well as encouraging glycoprotein synthesis, folding and assembly into properly functioning higher ordered complexes, N-glycosylation also provides Archaea with a strategy to cope with changing environments. Archaea can, moreover, exploit the apparent species-specific nature of N-glycosylation for selectivity in mating, and hence, to maintain species boundaries, and in other events where cell-selective interactions are required. At the same time, addressing components of N-glycosylation pathways across archaeal phylogeny offers support for the concept of an archaeal origin for eukaryotes. In this MicroReview, these and other recent discoveries related to N-glycosylation in Archaea are considered.

CRISPRi as an efficient tool for gene repression in archaea.

In the years following its discovery and characterization, the CRISPR-Cas system has been modified and converted into a multitude of applications for eukaryotes and bacteria, such as genome editing and gene regulation. Since no such method has been available for archaea, we developed a tool for gene repression in the haloarchaeon Haloferax volcanii by repurposing its endogenous type I-B CRISPR-Cas system. Here, we present the two possible approaches for gene repression as well as our workflow to achieve and assess gene knockdown, offer recommendations on protospacer selection and give some examples of genes we have successfully silenced.

Origins of major archaeal clades correspond to gene acquisitions from bacteria.

The mechanisms that underlie the origin of major prokaryotic groups are poorly understood. In principle, the origin of both species and higher taxa among prokaryotes should entail similar mechanisms--ecological interactions with the environment paired with natural genetic variation involving lineage-specific gene innovations and lineage-specific gene acquisitions. To investigate the origin of higher taxa in archaea, we have determined gene distributions and gene phylogenies for the 267,568 protein-coding genes of 134 sequenced archaeal genomes in the context of their homologues from 1,847 reference bacterial genomes. Archaeal-specific gene families define 13 traditionally recognized archaeal higher taxa in our sample. Here we report that the origins of these 13 groups unexpectedly correspond to 2,264 group-specific gene acquisitions from bacteria. Interdomain gene transfer is highly asymmetric, transfers from bacteria to archaea are more than fivefold more frequent than vice versa. Gene transfers identified at major evolutionary transitions among prokaryotes specifically implicate gene acquisitions for metabolic functions from bacteria as key innovations in the origin of higher archaeal taxa.

A CRISPRi-dCas9 System for Archaea and Its Use To Examine Gene Function during Nitrogen Fixation by Methanosarcina acetivorans.

CRISPR-based systems are emerging as the premier method to manipulate many cellular processes. In this study, a simple and efficient CRISPR interference (CRISPRi) system for targeted gene repression in archaea was developed. The Methanosarcina acetivorans CRISPR-Cas9 system was repurposed by replacing Cas9 with the catalytically dead Cas9 (dCas9) to generate a CRISPRi-dCas9 system for targeted gene repression. To test the utility of the system, genes involved in nitrogen (N2) fixation were targeted for dCas9-mediated repression. First, the nif operon (nifHI1I2DKEN) that encodes molybdenum nitrogenase was targeted by separate guide RNAs (gRNAs), one targeting the promoter and the other targeting nifD Remarkably, growth of M. acetivorans with N2 was abolished by dCas9-mediated repression of the nif operon with each gRNA. The abundance of nif transcripts was >90% reduced in both strains expressing the gRNAs, and NifD was not detected in cell lysate. Next, we targeted NifB, which is required for nitrogenase cofactor biogenesis. Expression of a gRNA targeting the coding sequence of NifB decreased nifB transcript abundance >85% and impaired but did not abolish growth of M. acetivorans with N2 Finally, to ascertain the ability to study gene regulation using CRISPRi-dCas9, nrpR1, encoding a subunit of the repressor of the nif operon, was targeted. The nrpR1 repression strain grew normally with N2 but had increased nif operon transcript abundance, consistent with NrpR1 acting as a repressor. These results highlight the utility of the system, whereby a single gRNA when expressed with dCas9 can block transcription of targeted genes and operons in M. acetivoransIMPORTANCE Genetic tools are needed to understand and manipulate the biology of archaea, which serve critical roles in the biosphere. Methanogenic archaea (methanogens) are essential for the biological production of methane, an intermediate in the global carbon cycle, an important greenhouse gas, and a biofuel. The CRISPRi-dCas9 system in the model methanogen Methanosarcina acetivorans is, to our knowledge, the first Cas9-based CRISPR interference system in archaea. Results demonstrate that the system is remarkably efficient in targeted gene repression and provide new insight into nitrogen fixation by methanogens, the only archaea with nitrogenase. Overall, the CRISPRi-dCas9 system provides a simple, yet powerful, genetic tool to control the expression of target genes and operons in methanogens.

Recruitment of CRISPR-Cas systems by Tn7-like transposons.

A survey of bacterial and archaeal genomes shows that many Tn7-like transposons contain minimal type I-F CRISPR-Cas systems that consist of fused cas8f and cas5f, cas7f, and cas6f genes and a short CRISPR array. Several small groups of Tn7-like transposons encompass similarly truncated type I-B CRISPR-Cas. This minimal gene complement of the transposon-associated CRISPR-Cas systems implies that they are competent for pre-CRISPR RNA (precrRNA) processing yielding mature crRNAs and target binding but not target cleavage that is required for interference. Phylogenetic analysis demonstrates that evolution of the CRISPR-Cas-containing transposons included a single, ancestral capture of a type I-F locus and two independent instances of type I-B loci capture. We show that the transposon-associated CRISPR arrays contain spacers homologous to plasmid and temperate phage sequences and, in some cases, chromosomal sequences adjacent to the transposon. We hypothesize that the transposon-encoded CRISPR-Cas systems generate displacement (R-loops) in the cognate DNA sites, targeting the transposon to these sites and thus facilitating their spread via plasmids and phages. These findings suggest the existence of RNA-guided transposition and fit the guns-for-hire concept whereby mobile genetic elements capture host defense systems and repurpose them for different stages in the life cycle of the element.

Diverse anaerobic methane- and multi-carbon alkane-metabolizing archaea coexist and show activity in Guaymas Basin hydrothermal sediment.

Anaerobic oxidation of methane greatly contributes to global carbon cycling, yet the anaerobic oxidation of non-methane alkanes by archaea was only recently detected in lab enrichments. The distribution and activity of these archaea in natural environments are not yet reported and understood. Here, a combination of metagenomic and metatranscriptomic approaches was utilized to understand the ecological roles and metabolic potentials of methyl-coenzyme M reductase (MCR)-based alkane oxidizing (MAO) archaea in Guaymas Basin sediments. Diverse MAO archaea, including multi-carbon alkane oxidizer Ca. Syntrophoarchaeum spp., anaerobic methane oxidizing archaea ANME-1 and ANME-2c as well as sulfate-reducing bacteria HotSeep-1 and Seep-SRB2 that potentially involved in MAO processes, coexisted and showed activity in Guaymas Basin sediments. High-quality genomic bins of Ca. Syntrophoarchaeum spp., ANME-1 and ANME-2c were retrieved. They all contain and expressed mcr genes and genes in Wood-Ljungdahl pathway for the complete oxidation from alkane to CO2 in local environment, while Ca. Syntrophoarchaeum spp. also possess beta-oxidation genes for multi-carbon alkane degradation. A global survey of potential multi-carbon alkane metabolism archaea shows that they are usually present in organic rich environments but are not limit to hydrothermal or marine ecosystems. Our study provided new insights into ecological and metabolic potentials of MAO archaea in natural environments.

Induction of a Toxin-Antitoxin Gene Cassette under High Hydrostatic Pressure Enables Markerless Gene Disruption in the Hyperthermophilic Archaeon Pyrococcus yayanosii.

The discovery of hyperthermophiles has dramatically changed our understanding of the habitats in which life can thrive. However, the extreme high temperatures in which these organisms live have severely restricted the development of genetic tools. The archaeon Pyrococcus yayanosii A1 is a strictly anaerobic and piezophilic hyperthermophile that is an ideal model for studies of extreme environmental adaptation. In the present study, we identified a high hydrostatic pressure (HHP)-inducible promoter (P hhp ) that controls target gene expression under HHP. We developed an HHP-inducible toxin-antitoxin cassette (HHP-TAC) containing (i) a counterselectable marker in which a gene encoding a putative toxin (virulence-associated protein C [PF0776 {VapC}]) controlled by the HHP-inducible promoter was used in conjunction with the gene encoding antitoxin PF0775 (VapB), which was fused to a constitutive promoter (P hmtB ), and (ii) a positive marker with the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase-encoding gene from P. furiosus controlled by the constitutive promoter P gdh The HHP-TAC was constructed to realize markerless gene disruption directly in P. yayanosii A1 in rich medium. The pop-out recombination step was performed using an HHP-inducible method. As proof, the PYCH_13690 gene, which encodes a 4-α-glucanotransferase, was successfully deleted from the strain P. yayanosii A1. The results showed that the capacity for starch hydrolysis in the Δ1369 mutant decreased dramatically compared to that in the wild-type strain. The inducible toxin-antitoxin system developed in this study greatly increases the genetic tools available for use in hyperthermophiles.IMPORTANCE Genetic manipulations in hyperthermophiles have been studied for over 20 years. However, the extremely high temperatures under which these organisms grow have limited the development of genetic tools. In this study, an HHP-inducible promoter was used to control the expression of a toxin. Compared to sugar-inducible and cold-shock-inducible promoters, the HHP-inducible promoter rarely has negative effects on the overall physiology and central metabolism of microorganisms, especially piezophilic hyperthermophiles. Previous studies have used auxotrophic strains as hosts, which may interfere with studies of adaptation and metabolism. Using an inducible toxin-antitoxin (TA) system as a counterselectable marker enables the generation of a markerless gene disruption strain without the use of auxotrophic mutants and counterselection with 5-fluoroorotic acid. TA systems are widely distributed in bacteria and archaea and can be used to overcome the limitations of high growth temperatures and dramatically extend the selectivity of genetic tools in hyperthermophiles.

Spatial-Temporal Pattern of Sulfate-Dependent Anaerobic Methane Oxidation in an Intertidal Zone of the East China Sea.

Methane is a primary greenhouse gas which is responsible for global warming. The sulfate-dependent anaerobic methane oxidation (S-AOM) process catalyzed by anaerobic methanotrophic (ANME) archaea and sulfate-reducing bacteria (SRB) is a vital link connecting the global carbon and sulfur cycles, and it is considered to be the overriding methane sink in marine ecosystem. However, there have been few studies regarding the role of S-AOM process and the distribution of ANME archaea in intertidal ecosystem. The intertidal zone is a buffer zone between sea and land and plays an important role in global geochemical cycle. In the present study, the abundance, potential methane oxidation rate, and community structure of ANME archaea in the intertidal zone were studied by quantitative PCR, stable isotope tracing method and high-throughput sequencing. The results showed that the potential S-AOM activity ranged from 0 to 0.77 nmol 13CO2 g-1 (dry sediment) day-1 The copy number of 16S rRNA gene of ANME archaea reached 106 ∼ 107 copies g-1 (dry sediment). The average contribution of S-AOM to total anaerobic methane oxidation was up to 34.5%, while denitrifying anaerobic methane oxidation accounted for the rest, which implied that S-AOM process was an essential methane sink that cannot be overlooked in intertidal ecosystem. The simulated column experiments also indicated that ANME archaea were sensitive to oxygen and preferred anaerobic environmental conditions. This study will help us gain a better understanding of the global carbon-sulfur cycle and greenhouse gas emission reduction and introduce a new perspective into the enrichment of ANME archaea.IMPORTANCE The sulfate-dependent anaerobic methane oxidation (S-AOM) process catalyzed by anaerobic methanotrophic (ANME) archaea and sulfate-reducing bacteria (SRB) is a vital link connecting the global carbon and sulfur cycles. We conducted a research into the spatial-temporal pattern of S-AOM process and the distribution of ANME archaea in coastal sediments collected from the intertidal zone. The results implied that S-AOM process was a methane sink that cannot be overlooked in the intertidal ecosystem. We also found that ANME archaea were sensitive to oxygen and preferred anaerobic environmental conditions. This study will help us gain a better understanding of the global carbon-sulfur cycle and greenhouse gas emission reduction and introduce a new perspective into the enrichment of ANME archaea.

Factors driving the distribution and role of AOA and AOB in Phragmites communis rhizosphere in riparian zone.

Ammonia oxidation, mainly driven by ammonia-oxidizing archaea (AOA) and bacteria (AOB), plays an important role in determining the rate of nitrification in riparian zones. However, the underlying factors driving the distribution and activity of AOA and AOB in riparian zones, especially in the rhizosphere of Phragmites communis remain unknown. This study revealed the dominance of AOA in ammonium oxidization with higher abundance and activity in both rhizosphere and bulk soil in summer and winter over AOB in riparian zones, based on molecular methods and double-inhibitors method. Phylogenetic analysis showed that 54d9 cluster and Nitrososphaera dominated the AOA community and Nitrosospira dominated the AOB, respectively. For the distribution of AOA and AOB, it was the spatial heterogeneity of physicochemical properties that had the most significant effect. Specifically, TOM & TC were the main physicochemical variables accounting for the difference in abundance and community composition of AOA, and TN had an important influence on AOB in the sediment/soil in riparian zones. For abundance and activity, seasonal heterogeneity and P. communis rhizosphere had a significant impact on the archaeal activity and abundance, respectively, but did not show significant influencing on AOB. These findings suggest that the small-scale environmental heterogeneities in riparian zones are important in shaping the community composition and abundance of AOA and AOB.

Plasticity of archaeal C/D box sRNA biogenesis.

Archaeal and eukaryotic organisms contain sets of C/D box s(no)RNAs with guide sequences that determine ribose 2'-O-methylation sites of target RNAs. The composition of these C/D box sRNA sets is highly variable between organisms and results in varying RNA modification patterns which are important for ribosomal RNA folding and stability. Little is known about the genomic organization of C/D box sRNA genes in archaea. Here, we aimed to obtain first insights into the biogenesis of these archaeal C/D box sRNAs and analyzed the genetic context of more than 300 archaeal sRNA genes. We found that the majority of these genes do not possess independent promoters but are rather located at positions that allow for co-transcription with neighboring genes and their start or stop codons were frequently incorporated into the conserved boxC and D motifs. The biogenesis of plasmid-encoded C/D box sRNA variants was analyzed in vivo in Sulfolobus acidocaldarius. It was found that C/D box sRNA maturation occurs independent of their genetic context and relies solely on the presence of intact RNA kink-turn structures. The observed plasticity of C/D box sRNA biogenesis is suggested to enable their accelerated evolution and, consequently, allow for adjustments of the RNA modification landscape.

Microhomology-Mediated High-Throughput Gene Inactivation Strategy for the Hyperthermophilic Crenarchaeon Sulfolobus islandicus.

Sulfolobus islandicus is rapidly emerging as a model system for studying the biology and evolution within the TACK lineage of the archaeal domain. As the tree of life grows, identifying the cellular functions of genes within this lineage will have significant impacts on our understanding of the evolution of the last archaeal eukaryote common ancestor (LEACA) and the differentiation of archaea from eukaryotes during the evolution of the modern-day cell. To increase our understanding of this key archaeal organism, we report a novel high-throughput method for targeted gene inactivation in S. islandicus through one-step microhomology-directed homologous recombination (HR). We validated the efficacy of this approach by systematically deleting 21 individual toxin-antitoxin gene pairs and its application to delete chromosomal regions as large as 50 kb. Sequence analysis of 96 ArgD+ transformants showed that S. islandicus can effectively incorporate donor markers as short segments through HR in a continuous or discontinuous manner. We determined that the minimal size of homology allowing native argD marker replacement was as few as 10 bp, whereas argD marker replacement was frequently observed when increasing the size of homology to 30 to 50 bp. The microhomology-mediated gene inactivation system developed here will greatly facilitate isolation of S. islandicus gene deletion strains, making generation of a collection of genome-wide targeted mutants feasible and providing a tool to investigate homologous recombination in this organism.IMPORTANCE Current procedures for the construction of deletion mutants of S. islandicus are still tedious and time-consuming. We developed a novel procedure based on microhomology-mediated HR, allowing for rapid and efficient removal for genetic regions as large as 50 kb. Our work will greatly facilitate functional genomic studies in this promising model organism. Additionally, we developed a quantitative genetic assay to measure HR properties in S. islandicus, providing evidence that the ability to incorporate short, mismatched donor DNA into the genome through HR was probably a common trait for members of the Sulfolobus genus that are recombinogenic.

Improvement of ST0452 N-Acetylglucosamine-1-Phosphate Uridyltransferase Activity by the Cooperative Effect of Two Single Mutations Identified through Structure-Based Protein Engineering.

We showed previously that the Y97N mutant of the ST0452 protein, isolated from Sulfolobus tokodaii, exhibited over 4 times higher N-acetylglucosamine-1-phosphate (GlcNAc-1-P) uridyltransferase (UTase) activity, compared with that of the wild-type ST0452 protein. We determined the three-dimensional structure of the Y97N protein to explore the detailed mechanism underlying this increased activity. The overall structure was almost identical to that of the wild-type ST0452 protein (PDB ID 2GGO), with residue 97 (Asn) interacting with the O-5 atom of N-acetylglucosamine (GlcNAc) in the complex without metal ions. The same interaction was observed for Escherichia coli GlmU in the absence of metal ions. These observations indicated that the three-dimensional structure of the Y97N protein was not changed by this substitution but the interactions with the substrate were slightly modified, which might cause the activity to increase. The crystal structure of the Y97N protein also showed that positions 146 (Glu) and 80 (Thr) formed interactions with GlcNAc, and an engineering strategy was applied to these residues to increase activity. All proteins substituted at position 146 had drastically decreased activities, whereas several proteins substituted at position 80 showed higher GlcNAc-1-P UTase activity, compared to that of the wild-type protein. The substituted amino acids at positions 80 and 97 might result in optimized interactions with the substrate; therefore, we predicted that the combination of these two substitutions might cooperatively increase GlcNAc-1-P UTase activity. Of the four double mutant ST0452 proteins generated, T80S/Y97N showed 6.5-times-higher activity, compared to that of the wild-type ST0452 protein, revealing that these two substituted residues functioned cooperatively to increase GlcNAc-1-P UTase activity.IMPORTANCE We demonstrated that the enzymatic activity of a thermostable protein was over 4 times higher than that of the wild-type protein following substitution of a single amino acid, without affecting its thermostability. The three-dimensional structure of the improved mutant protein complexed with substrate was determined. The same overall structure and interaction between the substituted residue and the GlcNAc substrate as observed in the well-characterized bacterial enzyme suggested that the substitution of Tyr at position 97 by Asn might slightly change the interaction. This subtle change in the interaction might potentially increase the GlcNAc-1-P UTase activity of the mutant protein. These observations indicated that a drastic change in the structure of a natural thermostable enzyme is not necessary to increase its activity; a subtle change in the interaction with the substrate might be sufficient. Cooperative effects were observed in the appropriate double mutant protein. This work provides useful information for the future engineering of natural enzymes.

Phylogenetic diversity and distribution of bacterial and archaeal amoA genes in the East China Sea during spring.

Microbial nitrification is a key process in the nitrogen cycle in the continental shelf ecosystems. The genotype compositions and abundance of the ammonia monooxygenase gene, amoA, derived from ammonia-oxidizing archaea (AOA) and bacteria (AOB) in two size fractions (2-10 and 0.2-2 µm), were investigated in the East China Sea (ECS) in May 2008 using PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR). Four sites were selected across the continental shelf edge: continental shelf water (CSW), Kuroshio branch water (KBW), transition between CSW and KBW (TCSKB) and coastal KBW (CKBW). The gene copy numbers of AOA-amoA were higher than those of AOB-amoA in ECS. The relative abundance of amoA to the total 16S rRNA gene level reached approximately 15% in KBW and CKBW for the free-living fraction of AOA, whereas the level was less than 0.01% throughout ECS for the AOB. A cluster analysis of the AOA-amoA-DGGE band pattern showed distinct genotype compositions in CSW in both the size fractions and in the surface of the TCSKB and KBW. Sequences of the DGGE bands were assigned to two clades. One of the clades exclusively consisted of sequences derived from the 2-10-µm fraction. This study revealed that AOA-amoA abundance dominated over AOB-amoA throughout the ECS, whereas the genotype composition of AOA-amoA were distributed heterogeneously across the water masses. Additionally, this is the first report showing the distribution of AOA-amoA genotypes characteristic to particle-associated AOA in the offshore of the East China Sea.

Transcription of mcrA Gene Decreases Upon Prolonged Non-flooding Period in a Methanogenic Archaeal Community of a Paddy-Upland Rotational Field Soil.

Methanogenic archaea survive under aerated soil conditions in paddy fields, and their community is stable under these conditions. Changes in the abundance and composition of an active community of methanogenic archaea were assessed by analyzing mcrA gene (encoding α subunit of methyl-coenzyme M reductase) and transcripts during a prolonged drained period in a paddy-upland rotational field. Paddy rice (Oryza sativa L.) was planted in the flooded field and rotated with soybean (Glycine max [L.] Merr.) under upland soil conditions. Soil samples were collected from the rotational plot in the first year, with paddy rice, and in the two successive years, with soybean, at six time points, before seeding, during cultivation, and after harvest as well as from a consecutive paddy (control) plot. By the time that soybean was grown in the second year, the methanogenic archaeal community in the rotational plot maintained high mcrA transcript levels, comparable with those of the control plot community, but the levels drastically decreased by over three orders of magnitude after 2 years of upland conversion. The composition of active methanogenic archaeal communities that survived upland conversion in the rotational plot was similar to that of the active community in the control plot. These results revealed that mcrA gene transcription of methanogenic archaeal community in the rotational field was affected by a prolonged non-flooding period, longer than 1 year, indicating that unknown mechanisms maintain the stability of methanogenic archaeal community in paddy fields last up to 1 year after the onset of drainage.

A Metagenome-Based Investigation of Gene Relationships for Non-Substrate-Associated Microbial Phosphorus Cycling in the Water Column of Streams and Rivers.

Phosphorus (P) is a nutrient of primary importance in all living systems, and it is especially important in streams and rivers which are sensitive to anthropogenic P inputs and eutrophication. Microbes are accepted as the primary mineralizers and solubilizers of P improving bioavailability for organisms at all trophic levels. Here, we use a genomics approach with metagenome sequencing of 24 temperate streams and rivers representing a total P (TP) gradient to identify relationships between functional genes, functional gene groupings, P, and organisms within the P biogeochemical cycle. Combining information from network analyses, functional groupings, and system P levels, we have constructed a System Relational Overview of Gene Groupings (SROGG) which is a cohesive system level representation of P cycle gene and nutrient relationships. Using SROGG analysis in concert with other statistical approaches, we found that the compositional makeup of P cycle genes is strongly correlated to environmental P whereas absolute abundance of P genes shows no significant correlation to environmental P. We also found orthophosphate (PO43-) to be the dominant factor correlating with system P cycle gene composition with little evidence of a strong organic phosphorous correlation present even in more oligotrophic streams.

Tide as Steering Factor in Structuring Archaeal and Bacterial Ammonia-Oxidizing Communities in Mangrove Forest Soils Dominated by Avicennia germinans and Rhizophora mangle.

Mangrove species are adapted to grow at specific zones in a tidal gradient. Here we tested the hypothesis that the archaeal and bacterial ammonia-oxidizing microbial communities differ in soils dominated by the mangrove species Avicennia germinans and Rhizophora mangle. Two of the sampling locations were tidal locations, while the other location was impounded. Differences in the community compositions of ammonia-oxidizing archaea (AOA) and bacteria (AOB) were analyzed by denaturing gradient gel electrophoresis (DGGE) of amoA genes and by MiSeq 16S rRNA gene-sequencing. The abundances of AOA and AOB were established by quantitative PCR of amoA genes. In addition, we analyzed the total microbial community composition based on 16S rRNA genes and explored the influence of soil physicochemical properties underneath Avicennia germinans and Rhizophora mangle on microbial communities. AOA were always more abundant than AOB, but the effect of mangrove species on total numbers of ammonia oxidizers was location-specific. The microbial communities including the ammonia oxidizers in soils associated with A. germinans and R. mangle differed only at the tidal locations. In conclusion, potential site-specific effects of mangrove species on soil microbial communities including those of the AOA and AOB are apparently overruled by the absence or presence of tide.

Microbial community of nitrogen cycle-related genes in aquatic plant rhizospheres of Lake Liangzi in winter.

This study investigated the community structure of ammonia-oxidizing bacteria /archaea (AOB and AOA), as well as the effects of four aquatic plants (namely Ceratophyllum demersum, Hydrilla verticillata, Potamogeton crispus, and Nymphaea tetragona) rhizospheres on the abundance of AOB amoA, AOA amoA, anammox 16S rRNA, nirK, and nirS in Lake Liangzi, China. Phylogenetic analysis revealed that most AOB groups were Nitrosospira and Nitrosomonas, in which Nitrosospira was dominant. The AOA amoA were affiliated with two branches of classical sequences which belonging to Thaumarchaeota: water/sediments branch and soil/sediments branch. The abundance of AOA amoA in the rhizospheres of aquatic plants were higher than in the non-rhizosphere (p < 0.05), indicating that aquatic plants may promote the growth of AOA. However, the anammox 16S rRNA showed the opposite trend relative to AOA amoA (p < 0.05). Redundancy analysis (RDA) showed that the differences in abundance of AOB, AOA, anammox bacteria, and denitrifying bacteria are very likely related to the different contents of ammonia nitrogen (NH4 + -N), pH and dissolved oxygen (DO) and thus to the rhizosphere states of aquatic plants.

The first Taxus rhizosphere microbiome revealed by shotgun metagenomic sequencing.

In the present study, the shotgun high throughput metagenomic sequencing was implemented to globally capture the features of Taxus rhizosphere microbiome. Total reads could be assigned to 6925 species belonging to 113 bacteria phyla and 301 species of nine fungi phyla. For archaea and virus, 263 and 134 species were for the first time identified, respectively. More than 720,000 Unigenes were identified by clean reads assembly. The top five assigned phyla were Actinobacteria (363,941 Unigenes), Proteobacteria (182,053), Acidobacteria (44,527), Ascomycota (fungi; 18,267), and Chloroflexi (15,539). KEGG analysis predicted numerous functional genes; 7101 Unigenes belong to "Xenobiotics biodegradation and metabolism." A total of 12,040 Unigenes involved in defense mechanisms (e.g., xenobiotic metabolism) were annotated by eggNOG. Talaromyces addition could influence not only the diversity and structure of microbial communities of Taxus rhizosphere, but also the relative abundance of functional genes, including metabolic genes, antibiotic resistant genes, and genes involved in pathogen-host interaction, bacterial virulence, and bacterial secretion system. The structure and function of rhizosphere microbiome could be sensitive to non-native microbe addition, which could impact on the pollutant degradation. This study, complementary to the amplicon sequencing, more objectively reflects the native microbiome of Taxus rhizosphere and its response to environmental pressure, and lays a foundation for potential combination of phytoremediation and bioaugmentation.

Diversity and seasonal distribution of ammonia-oxidizing archaea in the water column of a tropical estuary along the southeast Arabian Sea.

Diversity and distribution pattern of ammonia-oxidizing archaea (AOA) were studied across a salinity gradient in the water column of Cochin Estuary (CE), a tropical monsoonal estuary along the southeast Arabian Sea. The water column of CE was found to be nutrient rich with high bacterial (3.7-6.7 × 108 cells L-1) and archaeal abundance (1.9-4.5 × 108 cells L-1). Diversity and seasonal variation in the distribution pattern of AOA were studied using clone library analysis and Denaturing gradient gel electrophoresis (DGGE). Clone library analysis of both the amoA and 16S rRNA gene sequences showed similar diversity pattern, however the diversity was more clear when the 16S rRNA gene sequences were analyzed. More than 70% of the sequences retrieved were clustered under uncultured Thaumarchaeota group 1 lineage and the major fractions of the remaining sequences were grouped into the Nitrosopumilus lineage and Nitrosopelagicus lineage. The AOA community in the CE was less adaptable to changing environmental conditions and its distribution showed seasonal variations within the DGGE banding pattern with higher diversity during the pre-monsoon period. The distribution of AOA also showed its preference to intermediate salinity for their higher diversity. Summer monsoon associated runoff and flushing played a critical role in regulating the seasonality of AOA distribution.

Rapid evolutionary innovation during an Archaean genetic expansion.

The natural history of Precambrian life is still unknown because of the rarity of microbial fossils and biomarkers. However, the composition of modern-day genomes may bear imprints of ancient biogeochemical events. Here we use an explicit model of macroevolution including gene birth, transfer, duplication and loss events to map the evolutionary history of 3,983 gene families across the three domains of life onto a geological timeline. Surprisingly, we find that a brief period of genetic innovation during the Archaean eon, which coincides with a rapid diversification of bacterial lineages, gave rise to 27% of major modern gene families. A functional analysis of genes born during this Archaean expansion reveals that they are likely to be involved in electron-transport and respiratory pathways. Genes arising after this expansion show increasing use of molecular oxygen (P = 3.4 × 10(-8)) and redox-sensitive transition metals and compounds, which is consistent with an increasingly oxygenating biosphere.